1. Field of the Invention
The invention relates to a method of generating soluble extracellular domains of human gonadotropin hormone receptors in functional form by expression and secretion in the baculovirus/insect cell system. The invention is also related to a recombinant baculovirus transfer vector carrying DNA encoding the soluble receptor extracellular domains, insect cells expressing and secreting the soluble receptor extracellular domains, and a method of purifying these same soluble receptor extracellular domains.
2. Description of the Background Art
Follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG), and thyroid stimulating hormone (TSH) are large heterodimeric glycoproteins having a molecular weight of approximately 28-38 kDa and composed of a common .alpha. subunit non-covalently bound to a specific .beta. subunit that confers receptor binding specificity. While FSH, LH, and CG are classified as gonadotropins, TSH is not. However, TSH is considered a gonadotropin-like hormone by virtue of sharing many common characteristics with the gonadotropins FSH, LH, and CG. This family of gonadotropin/gonadotropin-like glycoprotein hormones all interact selectively with specific receptors comprising a subclass in the superfamily of G protein-coupled receptors which display seven transmembrane spanning segments as part of their transmembrane domain.
Like other known families of G protein-coupled receptors, the glycoprotein hormone receptors have an extracellular domain and a cytoplasmic tail in addition to the distinctive transmembrane domain. However, the glycoprotein hormone receptor family is distinguished from other G protein-coupled receptors, such as rhodopsin, .beta.2-adrenergic receptors, etc., by the large size (&gt;300 amino acid residues) of its extracellular domain or "ectodomain," which also contains multiple cysteine residues and N-linked glycosylation sites. Upon activation by their respective hormone ligands, the glycoprotein hormone receptors stimulate an increase in adenylyl cyclase activity and elevated intracellular adenosine 3', 5'-cyclic phosphate (cAMP) levels (Verrier et al., Eur. J. Biochem., 74:243-252, 1977; Hunzicker-Dunn et al., in Luteinizing Hormone Action and Receptors, p. 57, 1985, CRC Press, Boca Raton, Fla.; Field, in The Thyroid, p. 288, 1986, JB Lippencott Co., Philadelphia, Pa).
The binding specificity of the glycoprotein hormone receptor and its hormone ligand appears to reside principally in the extracellular portion of the receptor. In studies with the LH receptor, variants of the LH receptor molecule lacking the transmembrane domain and the C-terminal regions have been found to bind LH with high specificity and affinity (Tsai-Morris et al., J. Biol. Chem. 265:19385-19388, 1990; Xie et al., J. Biol. Chem. 265:21411-21414, 1991). Binding specificity has also been found to be conferred by the extracellular domain in chimeric glycoprotein hormone receptors created by interchanging N-terminal portions that include the extracellular domain (Braun et al., EMBO J. 10:1885-1890, 1991; Nagayama et al., Biochem. Biophys. Res. Commun. 173:1150-1156, 1990 and Proc Natl. Acad. Sci. USA 88:902-905, 1991). Davis et al., Mol. Endocrinol. 9:159-170, (1995) recently reported that the expression of the extracellular domain of rat FSHR by itself is sufficient for high affinity interactions with human FSH.
Although the extracellular high affinity hormone-binding domain of the rat LH receptor has been shown to be expressed independently of the domain that anchors the receptor in the cell membrane, the expressed extracellular domain was found to be retained intracellularly in the human kidney and COS host cells (Xie et al., J. Biol. Chem. 265:21411-21414, 1990; Moyle et al., J. Biol. Chem 266:10807-10812, 1991; Ji et al., Endocrinology 128:2648-2650, 1991; Pajot-Augy et al., J. Mol. Endocrinol. 14:51-66, 1995) with the exception of one study (Tsai-Morris et al., J. Biol. Chem. 265:19385-19388, 1990). Tsai-Morris et al., supra, reportedly found a naturally occurring soluble variant of the LH receptor, which is believed to be a product of alternative splicing, being secreted extracellularly by COS-7 cells. However, when Moyle et al., supra, expressed this same variant in COS-7 cells, they were unable to reproduce the secretion of soluble modified LH receptor into the culture medium. Instead, they observed that the LHR analogs or variants lacking the transmembrane domain were predominantly retained inside the cell. Kolena et al., Endocrinol. Exp. 20:339-348 (1993) and Wimalasena et al., Endocrinol. 113:618-624 (1983) reported the existence of naturally occurring soluble LH/CG binding proteins which might be receptor fragments. Bernard et al., Mol. Cell. Endocrinol. 71:R1914 R23 (1990) reported that alternatively spliced mRNA encoding a truncated LH receptor was expressed in rat ovary which may be an explanation for the previously reported soluble LH receptors. In a separate study with a truncated form (346 N-terminal amino acids) of rat FSHR representing the extracellular domain, it was reported that this truncated receptor was not secreted from human embryonic kidney cells, but rather trapped intracellularly despite the presence of a signal peptide and the absence of any membrane anchoring regions (Davis et al., supra).
The extracellular domain of the LH receptor was efficiently expressed in E. coli as a truncated receptor which formed inclusion bodies that required refolding with a mild denaturant, guanidine-HCl, to generate a multimeric hormone binding-component soluble form of the receptor (Chen et al., Mol. Cell. Endocrinol. 91:35-41, 1993). As heterologous proteins are expressed in E. coli host cells in nonglycosylated form, the work of Chen and coworkers suggests that the N-terminal 1-294 amino acid sequence of the LH receptor appears to have all the necessary information required for proper folding and that carbohydrate moieties on the soluble receptor are not required for proper folding or hormone binding activity of the soluble receptor extracellular domain. In contrast, Davis et al., supra, concluded from the results of their studies on N-linked glycosylation of rat FSHR, that while N-linked rat FSHR carbohydrates do not directly provide a binding site for the hormone, they instead appear to be required for proper folding of nascent receptors into a hormone-binding competent configuration.
A number of research groups have tried to take advantage of the baculovirus/insect cell system to overexpress gonadotropin-like hormone receptors either in full length or as truncated soluble receptor forms. The baculovirus/insect cell expression system offers the advantages of having strong or moderately strong promoters available for the high level expression of a heterologous protein as well as being noninfectious to vertebrates. This helper virus-independent expression system has been used to express heterologous proteins from a variety of different eukaryotes, prokaryotes, and viruses at levels up to 25% of total insect cell protein.
Full length rat FSHR and human FSHR were reported to have been expressed in the baculovirus/insect cell system as functional receptors (Liu et al, Endocrinol. 135:682-691, 1994; Christophe et al., Biochem. Biophys. Res. Commun. 196:402-408, 1993). However, no provisions for secretion of the soluble extracellular form of the receptor into the culture media was described in either case. Similarly, EP 0614975 A1 relates to the purification and cloning of receptors for LH, CG, FSH, and TSH but only specifically discloses examples with rat luteal LH/CG receptor and rat testicular FSH receptor. Again, no provisions for the secretion of a soluble extracellular form of the receptor was disclosed.
Tilly et al. (Society For The Study of Reproduction, vol. 46, supplement 1, abstract #330, 1992) reported the expression of a hormone-binding extracellular domain of hFSHR which was retained intracellularly in the soluble cell fraction of human fetal kidney cells. No secretion of soluble hFSHR receptor was ever disclosed.
Seetharamaiah et al. (Autoimmunity 14:315-320, 1993) disclosed that the hTSHR-EC domain of amino acid residues 1-395, was expressed in a baculovirus/insect cell system and obtained from whole cell lysates with no secreted receptor being detected in the culture media as no sequence encoding a signal peptide was present in the gene construct. In a later study, Seetharamaiah et al. (Endocrinol. 134:549-554, 1994) also reported the expression of hTSHR-EC in Sf9 insect cells where the receptor was obtained as protein aggregates from whole cell extracts and solubilization with guanidine-HCl was required for proper refolding into a soluble hormone-binding competent configuration.
In contrast, Huang et al. (J. Mol. Endocrinol. 10:127-142, 1993) reported the expression in a baculovirus/insect cell system of hTSHR-EC which failed to bind TSH hormone. The gene sequence encoding hTSHR-EC included the endogenous human signal sequence. Very minute amounts of truncated receptor, detectable in SDS-PAGE only when labeled with .sup.35 S, were found to be secreted, with almost all of the hTSHR-EC produced being retained intracellularly.
To summarize previous reports describing expression of extracellular domain of gonadotropin or gonadotropin-like receptors, the expressed truncated receptor was found to be trapped intracellularly, frequently in a non-functional form that requires further purification and refolding with a mild denaturant in order to obtain soluble hormone-binding competent receptor. Even gene constructs that included a signal sequence to facilitate secretion in host cells were unsuccessful in achieving any significant secretion of soluble receptors, functionally hormone-binding or otherwise. Previously, it has not been possible to reproducibly demonstrate secretion of any soluble gonadotropin or gonadotropin-like receptor. A possible explanation for this failure to secrete soluble hormone-binding receptors may be due to soluble receptors being identified as misfolded proteins by the cell and retained for degradation (Klauser et al., Cell 62:611-614 (1990)).
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